Genetic Dissection of Cis-Elements in Spatio-temporal Control of DNA Replication

DNA replication in all Eukaryotes follows a defined temporal order termed replication-timing program (RT), which is coupled with the spatial separation of chromatin distribution inside the nucleus. Early or late replicating chromatin self-organizes in 3D into sub-nuclear compartments at the nucleus interior or proximity to nuclear lamina respectively. RT is also highly correlated with multiple other features of the genome including transcriptional activity, chromatin composition, and mutational landscape. The molecular mechanisms regulating RT and linking these events are unclear. To investigate the role of DNA sequences in RT regulation, I adopted two parallel approaches to test the sufficiency and necessity of specific DNA segments in these processes. In the first approach, I developed an extra-chromosomal vector system (E-BAC) to show that determinants for RT and A/B compartmentalization are genetically encoded in ~200kb DNA sequences. In the second approach involving CRISPR (clustered regularly interspaced short palindromic repeats) mediated genome-editing, I identified three “early replication control elements” (ERCEs) internal of the domain that act redundantly and interdependently to give rise to both early replication and A/B compartmentalization of a pluripotency associated domain in mouse embryonic stem cells. The three ERCEs and other ERCE-like elements form the strongest CTCF-independent interactions among each other, which could drive the formation of A/B compartments inside the nucleus. The ERCEs also display a combination of active chromatin features resembling promoters and/or enhancers. They are implicated in gene regulation possibly by mediating the formation of transcription factories. These findings underscore the genetic influence on controlling multiple cellular processes, and highlight the complexity of cis regulation from the linear genome. The discovery of cis regulatory elements offers mechanistic insight linking highly correlated genomic features/activities, and provides opportunities to further dissect their relationship from a 3D perspective. Deeper understanding of genome regulation will hopefully enable the manipulation of these processes in cell function and disease.